It is well known in the art that the majority of cytotoxic cancer chemotherapeutic agents act in a relatively indiscriminate manner. The effectiveness of agents such as cyclophosphamide, cis-platinum and adriamycin is a consequence of DNA alkylation, DNA-repair defects and elevated DNA topoisomerase II levels, respectively, in susceptible cancer cell types. However these features, though highly significant for the positive clinical outcomes sometimes seen with these agents, are thus required to be counter-balanced by their high toxicity and generation of resistance mechanisms (Current Opinion in Chemical Biology 2009, 13:345-353).
Alterations of signal transduction pathways leading to uncontrolled cellular proliferation, survival, invasion, and metastases are hallmarks of the carcinogenic process. Protein Kinases are considered to be key regulators of these signal transduction pathways. It is becoming increasingly evident that many of these aberrations converge on a few key pathways involved in cancer cell signal transduction, including the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and the Raf/mitogen-activated and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascades. Both these pathways play important roles in normal cellular physiology; the carcinogenic process exploits and uses these same pathways to convey constitutively active survival signals to the nucleus. Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival (Cancer Treatment Reviews 39 (2013) 935-946).
Hence, over the past decade the development of anti-cancer drugs has undergone crucial changes. Whereas conventional chemotherapy targets both normal and rapidly dividing cells, newer agents tend to exploit tumor-specific alterations in DNA or in signal transduction pathways.
One of the approach might be to target specific DNA sequences that may combine high target selectivity with the prospect of developing small-drug-like molecules. This entails the targeting of DNA sequence motifs that fold into four-stranded structures called G-quadruplexes. Accumulating evidence has suggested the existence of the G-quadruplex conformation for telomeric DNA sequences both in vitro and in vivo.
Telomeres are DNA-protein complex, which are non-coding highly repetitive sequences located at the 3′ end region of the chromosomes consisting of almost 200 nucleotides constituted by tandem repeats of hexanucleotide (TTAGGG)n and finishes as an over-hanging single strand that provide protection against gene erosion at cell divisions, chromosomal non-homologous end-joining and nuclease attacks. The telomeres have a vital role for life as because of wide functioning with telomere associated proteins, attachment to the nuclear matrix, and higher order chromatin structures (Collado et al., 2007; Campbell, 2012). Conservation of telomeric length is an important biological condition for cell growth (Engelhardt & Finke, 2001; Lange, 2009). Telomeric DNA in vertebrates consists of tandem repeats of hexanucleotide sequence, d(TTAGGG). The G-rich single stranded sequence at the 3′-end of telomeric, DNA can adopt varying tertiary structures including G-Quadruplexes. After each cell division, the telomere sequence gets shortened and that leads to halting of cell division (senescence) and eventually controlled cell death (apoptosis) takes place. Telomerase is the enzyme which functionalizes the addition of hexanucleotide repeats of TTAGGG to the 3′-end of telomere and it's maintenance during normal cell division (mitosis). Unusual over expression of telomerase enzyme engineers massive extension of telomeric ends and brings in anomalous cell proliferation, which causes cancer. Moreover, it has also been demonstrated that in 85% of cancer cells telomerase is over expressed, which prevents natural shortening of telomere and leads to cell proliferation.
Telomeres and telomerase are thus also known to be attractive therapeutic targets in cancer because telomerase is found to be expressed in 80-85% of cancer cells and primary tumours, but not in normal somatic cells.
Telomeres are the main location which forms such crucial functional secondary structures of DNA comprising of a 3′-end region of chromosomes consists of almost 200 nucleotides constituted by tandem repeats of hexanucleotide (TTAGGG)n and finishes as an over-hanging single strand. The overhang G-rich repetitive DNA units at 3′-end of telomeres can form various tertiary structures including G-Quadruplex where the guanine bases stack over each other and were stabilized by cyclic Hoogesteen type of hydrogen-bonding (Dai & Carver., 2007; Luu et al., 2008; Burge et al., 2006; Dai & Punchihewa, 2007). The most promising fact of the G-Quadruplex structure is its topology, which cannot be recognized by the single-stranded RNA component of telomerase enzyme. On the contrary it can be recognized by itself as a damage signal of DNA and therefore can invoke apoptosis (Rodriguez et al., 2008).
Though inhibition of telomerase action is an efficient way to tune back cancer cells for natural cell death, one prominent emerging strategy for telomerase inhibition is the stabilization of G-Quadruplex structures of telomeric DNA, thus preventing its availability as a primer for telomerase assisted elongation whereby telomerase activity would be down regulated to bring in apoptosis in cancerous cells.
Quarfloxin (or Quarfloxacin) is known as a fluoroquinolone derivative with antineoplastic activity. Quarfloxin disrupts the interaction between the nucleolin protein and a G-quadruplex DNA structure in the ribosomal DNA (rDNA) template, a critical interaction for rRNA biogenesis that is overexpressed in cancer cells; disruption of this G-quadruplex DNA:protein interaction in aberrant rRNA biogenesis may result in the inhibition of ribosome synthesis and tumor cell apoptosis.
Only very few of the previously investigated compounds in the art have shown specific inhibition of telomerase using nanomolar concentration reflected in their IC50 values and therefore, there still remains a need in the art to explore for selective molecules that would have high binding selectivity towards the intramolecular G-Quadruplex structures of telomere over a DNA duplex structure or other potential quadruplex forming sequences in the genome that would effectively stabilize the said G-Quadruplex structures of telomere in minimum effective concentrations at which concentration or dose it would kill more than 50% of cancerous cells but not be cytotoxic to normal cells.
Quite interestingly, there is now a growing body of work that has explored a hypothesis that links the existence of G-quadruplex-forming sequences in promoters of oncogenes including that of kinases. Thus, discovery of Quadruplex stabilizers which bind to promoter regions of oncogenes thereby inhibiting their transcription is quite relevant to cancer therapeutics (Current Opinion in Chemical Biology 2009, 13:345-353).
Further, it is also known in the art that in more than 50% of the cancers, p53 (tumor suppressor protein) is mutated and many existing chemotherapeutic drugs are found to be ineffective in p53 mutated cancers and hence it is also a major challenge of the day to develop anti-cancer agents that can kill cancer cells with non-functional p53.